Antisense polynucleotides to induce exon skipping and methods of treating dystrophies

ABSTRACT

Antisense polynucleotides and their use in pharmaceutical compositions to induce exon skipping in targeted exons of the gamma sarcoglycan gene are provided, along with methods of preventing or treating dystrophic diseases such as Limb-Girdle Muscular Dystrophy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/426,348, filed Mar. 5, 2015, which is a U.S. National Phase ofPCT/US2013/058636, filed Sep. 6, 2013, which claims the priority benefitunder 35 U.S.C. §119(e) of Provisional U.S. Patent Application No.61/697,766, filed Sep. 6, 2012, the disclosures of which areincorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant NumberHL061322 and U54AR052646 awarded by the National Institutes of Health.The government has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to antisense polynucleotides and theiruse in pharmaceutical compositions to induce exon skipping in targetedexons of the gamma sarcoglycan gene (SGCG), useful in treating variousforms of Muscular Dystrophy.

BACKGROUND OF THE INVENTION

Muscular Dystrophy (MD) is a group of more than 30 genetic disorderscharacterized by skeletal muscle weakness and degeneration. Limb-girdleMuscular Dystrophy (LGMD) is an autosomal class of MD. The most severelyaffected muscles in LGMD are those of limbs, including arms and legs, aswell as the trunk muscles affecting posture and breathing. MD patientsare born with normal muscle function but develop heart failure andbreathing problems due to the loss of mass and strength of cardiac andrespiratory muscle. Limb Girdle Muscular Dystrophy type 2C (LGMD2C) iscaused by mutations in the γ-sarcoglycan (Sgcg) gene [Noguchi et al.,Science 270: 819-822 (1995); McNally et al., Am J Hum Genet 59:1040-1047 (1996); Lasa et al., Eur J Hum Genet 6: 396-399 (1998)].γ-sarcoglycan is a dystrophin-associated protein [Allikian et al.,Traffic 8: 177-183 (2007)]. Dystrophin is a large rod-shaped cytoplasmicprotein found along the inner surface of the plasma membrane of musclecells. Dystrophin is encoded by the DMD gene, which is the largest geneknown so far, spanning 2.4 MB on the X chromosome [Hoffman, et al.,Biotechnology 24: 457-466 (1992)]. Mutations in the DMD gene are themost common cause of muscular dystrophy, affecting 1 in every 3500newborn boys worldwide [Moser, Hum Genet 66: 17-40 (1984)].

The dystrophin complex localizes at the muscle membrane, known as thesarcolemma, and connects intracellular actin bundles to theextracellular matrix. The dystrophin complex plays a critical role instabilizing the sarcolemma during muscle contraction. Mutation or lossof dystrophin or the associated sarcoglycans leads to destabilization ofthe sarcolemma and subsequent events, such as muscle cell injury, musclecell necrosis and fibrotic or fatty tissue deposition.

Mice and humans have a highly conserved dystrophin complex. The Sgcgnull mouse model (Sgcg^(−/−)) was the first model established for LGMDby deleting exon 2 of Sgcg, resulting in a null allele [Hack et al., JCell Biol 142: 1279-1287 (1998)]. γ-sarcoglycan mutant mice developprogressive disease pathology that resembles that of LGMD2C patients.Sgcg^(−/−) mice are born in the expected Mendelian ratios. By 20 weeksof age, however, half of the Sgcg^(−/−) mice die and the surviving miceweigh significantly less than wild-type littermates. Dystrophic changesof skeletal muscle, such as broad variation in fiber size, immune cellinfiltration and fibrotic and fatty tissue deposition, are evident by 3weeks of age but become prominent around 8 weeks of age. Consistent withthe disease progression pattern in patients, cardiomyopathy inSgcg^(−/−) also develops at later stage. At 20 weeks of age, Sgcg^(−/−)hearts display remarkable fibrosis and reduced cardiac function.

Disruption of the dystrophin complex makes the muscle membrane morefragile and more susceptible to membrane tears when subject to theshearing force during contraction. As a result of these tears,dystrophin or sarcoglycan null skeletal muscles show increasedpermeability that allows soluble enzymes such as creatine kinase to exitfrom the cell and blood proteins such as albumin or ions such as calciumto enter the cell. Initially, the membrane repairing machinery,including dysferlin family proteins, is activated to reseal the damagedmembrane [Bansal Nature 423: 168-172 (2003); Doherty et al., Development132: 5565-5575 (2005)]. However, this blurring of cell-environmentboundary and increased cytoplasmic calcium content are associated with aseries of harmful cellular events, such as increased reactive oxygenspecies, activation of protease cascade, and eventually lead to necroticcell death [Goldstein et al., J Gen Physiol 136: 29-34 (2010)].

Loss of muscle fibers also activates muscle stem cells, called satellitecells. Satellite cells divide and attempt to repair injured musclefibers. As myoblasts only have limited dividing potential, thedegeneration trend gradually overcomes the regeneration efforts,resulting in irreversible muscle loss. The loss of muscle bulk isaccompanied by replacement of connective and fatty tissue. Like humans,mutant mice also develop cardiomyopathy as a result of loss ofcardiomyocytes and fibrotic tissue deposition. Hearts withcardiomyopathy fail to pump properly, which results in a failure todeliver oxygen and nutrients to the tissue.

Drosophila has a simplified dystrophin complex that shows conservationwith mammals. Drosophila γ/δ-sarcoglycan (Sgcd) is equally similar tomammalian γ-sarcoglycan and δ-sarcoglycan. Previous work resulted in thegeneration of a fly model of muscular dystrophy by inducing a largedeletion in the Sgcd locus via imprecise P-element excision [Allikian etal., Hum Mol Genet 16: 2933-2943 (2007)]. The Sgcd⁸⁴⁰ line was selectedand further characterized because it had a defined deletion that ablatesexpression of the single γ/δ-sarcoglycan gene. Similar to theprogressive nature of MD in mammals, Sgcd mutant flies are normal whenthey emerge as adults, but develop heart and skeletal muscleabnormalities over time. Unlike the four-chamber heart in mammals, flieshave a simple heart tube to promote the blood circulation. Compared towild-type flies, heart tubes in Sgcd mutant flies are enlarged anddefective in contraction. Weakness in skeletal muscle manifests in theimpaired climbing ability of the mutant flies. Histological examinationof flight muscle in mutant flies also revealed muscle fiber detachmentfrom the exoskeleton, which is only infrequently seen in wild-typeflies.

The most common causes of muscular dystrophy are mutations in thedystrophin gene. Different mutations in dystrophin lead to differentseverities. For example, a mutation which shifts the reading frame, suchas the deletion of exon 43 to exon 48 [Doriguzzi et al., Eur Neurol 33:454-460 (1993)], leads to a much more severe disease than one with alarger deletion spanning exon 13 to 48 [Passos-Bueno et al., Hum MolGenet 3: 919-922 (1994)]. The frame shift in the former case results inloss of the C-terminus of dystrophin, which is responsible for normallocalization of sarcoglycans and other dystrophin-associated proteins.Furthermore, the level of dystrophin protein is also greatly reduced,possibly due to nonsense-mediated decay or improper protein folding andsubsequent degradation. In the latter case, the large deletion reducesthe number of spectrin repeats in the middle region of the protein,while keeping the crucial C-terminus and N-terminus intact. Thissuggests that internally truncated protein can be partially functional.These milder mutations are known as forms of Becker Muscular Dystrophy(BMD).

Most eukaryotic genes are made of protein-coding exons and non-codingintrons. Splicing is required to connect exons to form mature mRNA. Toachieve a proper splicing pattern, recognition of splice donor, spliceacceptor and exonic splicing enhancer (ESE) sites by the splicingmachinery are required. Blocking essential splice sites by antisensepolynucleotides (AONs) induces exclusion of certain exons from themature mRNA [Aartsma-Rus et al., RNA 13: 1609-1624 (2007)]. This eventis referred to as exon skipping.

Phase I and phase II clinic trials of therapeutic exon skipping havebeen carried out, proving the safety of AON administration andefficiency of dystrophin restoration [Bertoni, Front Biosci 13: 517-527(2008); Cirak et al., Lancet 378: 595-605 (2011)]. In the phase IItrial, 19 patients aged from 5-15 participated in the study. They weredivided into multiple groups that received escalated doses of AVI-4568(the AON drug) via weekly intravenous infusion for 12 weeks. No seriousdrug-related adverse effect was observed. Targeted exon skipping wasobserved in all patients and new dystrophin production was detected in adose dependent manner. The 3 patients with greatest response to the drughad 15%, 21% and 55% dystrophin positive fibers. Consistent with thereproduction of functional dystrophin, dystrophin-associated proteinswere also found restored at the plasma membrane of muscle cells.

SUMMARY OF THE INVENTION

The disclosure is directed to one or more antisense polynucleotides andtheir use in pharmaceutical compositions in a strategy to induce exonskipping in the gamma sarcoglycan gene in patients suffering fromLimb-girdle Muscular Dystrophy-2C (i.e., LGMD2C) or in patients at riskof such a disease. The disclosure also provides methods of preventing ortreating muscular dystrophy, e.g., LGMD2C, by exon skipping in the gammasarcoglycan gene using antisense polynucleotides.

One aspect the disclosure provides an isolated antisense polynucleotidewherein the polynucleotide specifically hybridizes to an exon targetregion of a gamma sarcoglycan RNA, wherein the exon is selected from thegroup consisting of exon 4 (SEQ ID NO: 1), exon 5 (SEQ ID NO: 2), exon 6(SEQ ID NO: 3), exon 7 (SEQ ID NO: 4) and a combination thereof. In someembodiments, the antisense polynucleotide cannot form an RNase Hsubstrate, and in further embodiments the antisense polynucleotidecomprises a modified polynucleotide backbone. In some embodiments, themodified backbone is a 2′-O-methyl-oligoribonucleotide.

Exon (SEQ ID NO) Sequence (5′-3′) 4ATTTTGCAAATTTTATAAATCTCTTTCTAGGACTCATCTCTGC (SEQ IDTTCTACAATCAACCCAGAATGTGACTGTAAATGCGCGCAACT NO: 1)CAGAAGGGGAGGTCACAGGCAGGTTAAAAGTCGGTGAGTCC AGCTTCATCATGGTGCTTTGCA 5AGTTTATAATAAACTGTTTTAATTCTTCAGGTCCCAAAATGG (SEQ IDTAGAAGTCCAGAATCAACAGTTTCAGATCAACTCCAACGAC NO: 2)GGCAAGCCACTATTTACTGTAGATGAGAAGGAAGTTGTGGTTGGTACAGATAAACTTCGAGTAACTGGTATGTACTAACTCGAG AAAAACACAACAT 6GCTCCTGATACATCTTTGTTTTTTGTTTAGGGCCTGAAGGGGC (SEQ IDTCTTTTTGAACATTCAGTGGAGACACCCCTTGTCAGAGCCGA NO: 3)CCCGTTTCAAGACCTTAGGTAAGAATTTTTGTTCAAATATTA ACAACC 7ATTTTTAATACTTTTTTTTTTTTTTTTTGTGCTTCTTTTCCTC (SEQ IDATCTCAGATTAGAATCCCCCACTCGGAGTCTAAGCATGGATGC NO: 4)CCCAAGGGGTGTGCATATTCAAGCTCACGCTGGGAAAATTGAGGCGCTTTCTCAAATGGATATTCTTTTTCATAGTAGTGATGGAATGGTGAGTTCATTCACAGATCAGCCTCCTACT Underlining indicates coding regionof exon. Antisense polynucleotides are contemplated to be sequences thattarget an exon or an intron-exon boundary.

Table 1. Gamma Sarcoglycan Exon Sequences.

In further embodiments, the disclosure contemplates that the modifiedpolynucleotide backbone comprises a modified moiety substituted for atleast one sugar of at least one of the polynucleotides. In a specificembodiment, the modified moiety is a Morpholino.

It is additionally contemplated by the disclosure that, in someembodiments, the modified polynucleotide backbone of the polynucleotidecomprises at least one modified internucleotide linkage, and in someembodiments the modified internucleotide linkage comprises a modifiedphosphate. The modified phosphate, in various embodiments, is selectedfrom the group consisting of a methyl phosphonate, a methylphosphorothioate, a phosphoromorpholidate, a phosphoropiperazidate and aphosphoroamidate.

The disclosure also provides embodiments wherein the polynucleotidecomprises a peptide nucleic acid.

In still further embodiments, it is contemplated that the polynucleotideis chemically linked to one or more conjugates that enhance theactivity, cellular distribution, or cellular uptake of the antisensepolynucleotide. In related embodiments, the conjugate is a peptide thatenhances cellular uptake, and in further embodiments the peptide isselected from the group consisting of a nuclear localization signal(NLS), HIV-1 TAT protein, a peptide comprising an integrin bindingdomain, an oligolysine, an adenovirus fiber protein and a peptidecomprising a receptor-mediated endocytosis (RME) domain.

In some embodiments, the polynucleotide is chemically linked to apolyethylene glycol molecule.

In another aspect of the disclosure, a pharmaceutical composition isprovided comprising the antisense polynucleotide as described herein anda physiologically compatible phosphate buffer. In some embodiments, thepharmaceutical composition further comprises an additional antisensepolynucleotide, wherein the additional polynucleotide specificallyhybridizes to an exon of a gamma sarcoglycan nucleic acid.

A further aspect of the disclosure provides a method of inducingexon-skipping of a gamma sarcoglycan RNA, comprising delivering to acell a therapeutically effective or prophylactically effective amount ofthe antisense polynucleotide or a composition of the disclosure, therebyinducing exon-skipping of the gamma sarcoglycan RNA.

In some embodiments, the cell is a human muscle cell and in furtherembodiments the human muscle cell is in a patient. The patient, invarious embodiments, is a patient that has muscular dystrophy. Infurther embodiments, the muscular dystrophy is Limb Girdle MuscularDystrophy type 2C (LGMD2C).

In still another aspect of the disclosure, a method of ameliorating LimbGirdle Muscular Dystrophy type 2C (LGMD2C) in a patient in need thereofis provided, comprising the step of administering to the patient atherapeutically effective amount of a composition of the disclosure,thereby ameliorating LGMD2C.

The disclosure also provides a method of inhibiting the progression ofdystrophic pathology associated with LGMD2C in a patient in need thereofcomprising the step of administering to the patient a therapeuticallyeffective amount of a composition of the disclosure, thereby inhibitingthe progression of dystrophic pathology.

In some aspects, the disclosure provides a method of improving musclefunction in a patient suffering from Limb Girdle Muscular Dystrophy type2C (LGMD2C) comprising the step of administering to the patient atherapeutically effective amount of a composition of the disclosure,thereby improving muscle function. In some embodiments, the improvementis a cardiac muscle improvement, and in further embodiments theimprovement in muscle function is an improvement in muscle strength. Infurther embodiments, the improvement in muscle strength is animprovement in respiratory muscle strength, and in additionalembodiments the improvement in muscle function is an improvement inmotor stability. In any of the embodiments of the disclosure, theimprovement in respiratory muscle strength is measured as reducednocturnal desaturation events or improved pulmonary function testing. Inany of the embodiments of the disclosure, the improvement in motorstability results in an improved time to standing or climbing stairsrelative to previously measured time to standing or climbing stairs.

The improvement in motor stability, in some embodiments, results in animproved six-minute walk test by the patient relative to a previouslymeasured six-minute walk test by that patient. Additional improvementscontemplated by the disclosure are assessed via improved histopathologyor improved noninvasive imaging evidence such as reduced fibrofattyinfiltration and reduced evidence of scarring. Cardiac muscleimprovement as documented by improved left and right ventricularfunction, reduced right and left ventricular diameters and reducedevidence of cardiac damage is also contemplated.

Another aspect of the disclosure is drawn to a kit comprising theantisense polynucleotides as described herein, optionally in acontainer, and a package insert, package label, instructions or otherlabeling. In some embodiments, the kit further comprises an additionalpolynucleotide, wherein the additional polynucleotide specificallyhybridizes to an exon in a gamma sarcoglycan RNA.

Additional aspects and embodiments of the disclosure are described inthe following enumerated paragraphs.

1. An isolated polynucleotide encoding a mini-gamma sarcoglycan proteincomprising a deletion of an exon selected from the group consisting ofexon 4 (SEQ ID NO: 1), exon 5 (SEQ ID NO: 2), exon 6 (SEQ ID NO: 3),exon 7 (SEQ ID NO: 4) and a combination thereof.

2. A composition comprising a viral vector comprising the polynucleotideaccording to paragraph 1.

3. The composition of paragraph 2, wherein the viral vector is selectedfrom the group consisting of a herpesvirus and an adenovirus.

4. The composition of paragraph 2 or paragraph 3, wherein the viralvector is adeno-associated virus-9 (AAV-9).

5. The composition of any one of paragraphs 2-4, wherein the deletioncomprises exon 5.

6. The composition of any one of paragraphs 2-4, wherein the deletioncomprises exon 4, exon 5, exon 6 and exon 7.

7. The composition of any one of paragraphs 2-4, wherein the deletionconsists of exon 5.

8. The composition of any one of paragraphs 2-4, wherein the deletionconsists of exon 4, exon 5, exon 6 and exon 7.

9. An isolated mini-gamma sarcoglycan polypeptide, wherein the gammasarcoglycan comprises at least one deletion in an exon selected from thegroup consisting of exon 4, exon 5, exon 6 and exon 7.

10. The mini-gamma sarcoglycan polypeptide of paragraph 9 that comprisesa sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 8.

11. An isolated polypeptide that is at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 99% or 100% identical to the mini-gamma sarcoglycanpolypeptide of paragraph 9 or paragraph 10.

12. A pharmaceutical composition comprising the mini-gamma sarcoglycanpolypeptide of any one of paragraphs 9-11 and a pharmaceuticallyacceptable carrier, diluent, or excipient.

13. The composition of any one of paragraphs 2-8 or 12, furthercomprising an additional therapeutic agent.

14. The composition of paragraph 13, wherein the additional therapeuticagent is selected from the group consisting of a glucocorticoid steroid,an angiotensin converting enzyme inhibitor, a beta adrenergic receptorblocker, an anti-fibrotic agent and a combination thereof.

15. The composition of any one of paragraphs 2-8 or 12-14, furthercomprising a pharmaceutically acceptable carrier, diluent, or excipient.

16. A host cell comprising the polynucleotide of paragraph 1 or thecomposition of any one of paragraphs 2-8.

17. A method of expressing a mini-sarcoglycan protein in a cell, themethod comprising contacting the cell with the isolated polynucleotideof paragraph 1 or the composition of any one of paragraphs 2-8 or 13under conditions that result in expression of the mini-sarcoglycanprotein in the cell.

18. The method of paragraph 17, wherein the contacting occurs in vivo.

19. The method of paragraph 17 or paragraph 18, wherein the cell is amuscle cell.

20. The method of any one of paragraphs 17-19, wherein the cell is amammalian cell.

21. The method of paragraph 20, wherein the mammalian cell is a humancell.

22. The method of paragraph 21, wherein a human comprising the humancell has muscular dystrophy.

23. The method of paragraph 22, wherein the muscular dystrophy is LimbGirdle Muscular Dystrophy type 2C (LGMD2C).

24. The method of paragraph 17, wherein the contacting occurs in vitro.

25. The method of paragraph 24, wherein the cell is a muscle cell.

26. The method of any one of paragraphs 17 or 24-25, wherein the cell isa mammalian cell.

27. The method of paragraph 26, wherein the mammalian cell is a humancell.

28. The method of paragraph 27, wherein a human comprising the humancell has muscular dystrophy.

29. A method of ameliorating Limb Girdle Muscular Dystrophy type 2C(LGMD2C) in a patient in need thereof comprising the step ofadministering to the patient a therapeutically effective amount of thepolynucleotide of paragraph 1 or the composition of any one ofparagraphs 2-8 or 12-15, thereby ameliorating LGMD2C.

30. A method of inhibiting the progression of dystrophic pathologyassociated with LGMD2C in a patient in need thereof comprising the stepof administering to the patient a therapeutically effective amount ofthe polynucleotide of paragraph 1 or the composition of any one ofparagraphs 2-8 or 12-15, thereby inhibiting the progression ofdystrophic pathology.

31. A method of improving muscle function in a patient suffering fromLimb Girdle Muscular Dystrophy type 2C (LGMD2C) comprising the step ofadministering to the patient a therapeutically effective amount of thepolynucleotide of paragraph 1 or the composition of any one ofparagraphs 2-8 or 12-15, thereby improving muscle function.

32. The method of paragraph 31 wherein the improvement is a cardiacmuscle improvement.

33. The method of paragraph 31 or paragraph 32 wherein the improvementin muscle function is an improvement in muscle strength.

34. The method of paragraph 33 wherein the improvement in musclestrength is an improvement in respiratory muscle strength.

35. The method of paragraph 29 or paragraph 30 wherein the improvementin muscle function is an improvement in motor stability.

36. The method of paragraph 35 wherein the improvement in motorstability results in an improved six-minute walk test by the patientrelative to a previously measured six-minute walk test by that patient.

37. A kit comprising the polynucleotide of paragraph 1, the polypeptideof any one of paragraphs 9-11 or the composition of any one ofparagraphs 2-8 or 12, optionally in a container, and a package insert,package label, instructions or other labeling.

38. The kit of paragraph 37, further comprising an additionaltherapeutic agent.

39. The kit of paragraph 38, wherein the additional therapeutic agent isselected from the group consisting of a glucocorticoid steroid, anangiotensin converting enzyme inhibitor, a beta adrenergic receptorblocker, an anti-fibrotic agent and a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reading frame of the mammalian Sgcg gene before (A) andafter (C) skipping exon 4-7. Deletion of one thymine in exon 6 disruptsthe reading frame and is the most common LGMD2C mutation. (B). Skippingexon 4-7 restores the reading frame. Exons that follow exon “E8” andthose that are labeled “E1” represent untranslated regions.

FIG. 2 depicts the structure of full-length γ-sarcoglycan (left) and theinternally truncated γ-sarcoglycan after exon 4-7 is skipped (right).Conserved amino acids are shown.

FIG. 3 depicts vector maps of mini-Sgcg constructs in transgenic flies(A) and transgenic mice (B). UAS is the enhancer sequence specificallyrecognized by Gal4. The desmin (Des) promoter is muscle specific.Preliminary data has shown mini-Sgcg is produced in UAS-mini-Sgcgtransgenic flies and in Des-mini-Sgcg transfected muscle cells inculture. Note that mini-Sgcg produced from both transgenes is taggedwith the Xpress epitope tag.

FIG. 4 shows that the Drosophila heart tube is a thin-walled structurethat runs along the dorsum of the adult fly (A). B shows an EM image ofthe heart tube (h and arrows). A and B are from Curtis, Morphology 240:225 (1999). C shows the membrane localized staining from the mini-Sgcgtransgene expressing mini-Sgcg in Sgcg⁸⁴⁰ flies in the heart tube usinga Tinman GAL4.

FIG. 5 depicts that full-length Sgcg and mini-Sgcg are expressed andlocalize correctly to the plasma membrane of transgenic fly skeletalmuscle cells. Muscle cells are aligned parallel to each other.

FIG. 6 depicts improved heart function in mini-Sgcg rescued Sgcd⁸⁴⁰flies. Shown on the left is the End Systolic Dimension (ESD). Sgcd⁸⁴⁰flies have increased ESD, indicating heart tube dilation. Thisenlargement is rescued when mini-Sgcg protein is introduced into Sgcd⁸⁴⁰flies via transgenesis.

FIG. 7 shows the construct to produce mini-Sgcg in mice. A positivetransgenic founder mouse (#23, called “MJ”) is indicated.

FIG. 8 shows that mini-Sgcg protein is produced in cultured myotubeswhen transfected with the Des-mini-Sgcg vector and localizes to themuscle membrane.

FIG. 9 depicts the rescue of walking ability in Sgcd null flies usingmini-Sgcg. On the left panel, the Y axis is number of beam breaks perhour, and the X axis is hour where midnight is designated as 0. On theright panel, the Y axis is total number of beam breaks from midnight to8 AM.

FIG. 10 shows that expression of mini-Sgcg in Sgcd⁸⁴⁰ fliessignificantly improved nocturnal activity.

FIG. 11 shows that mini-Sgcg protein can be stably produced in mammalianskeletal muscle (skeletal) and heart (Heart).

FIG. 12 depicts mini-Sgcg expression in two different transgenic lines.The upper panel shows expression at three different concentrations fromtransgenic line 50 or transgenic line 84.

FIG. 13 depicts that mini-Sgcg protein localizes to the plasma membraneof skeletal muscle when expressed in wildtype normal mice (Tg⁺). Thissame signal was not detected in transgenic negative (Tg⁻) muscledemonstrating that this signal derives from the transgene.

FIG. 14 shows that expression of full length endogenous γ-sarcoglycanprotein (Sgcg) is diminished at the plasma membrane when mini-Sgcg ispresent (Tg⁺ (left panel) versus when mini-Sgcg is absent (Tg⁻ (rightpanel)).

FIG. 15 depicts a model for sarcoglycan assembly.

FIG. 16 shows that mini-Sgcg is enriched in the heavy microsomalfraction of muscle.

FIG. 17 shows two human cell lines with SGCG mutations that have beeninfected with retroviruses expressing telomerase and MyoD. The top rowis from an LGMD 2C patient whose disease arises from a mutation deletingexon 7 in SGCG. The bottom row is from an LGMD 2C patient who is deletedfor exon 6 of SGCG.

FIG. 18 depicts LGMD 2C cell lines differentiated into the musclelineage (6 days of differentiation). The top row is from an LGMD 2Cpatient whose disease arises from a mutation deleting exon 6 in SGCG,and the bottom row is from an LGMD 2C patient who is deleted for exon 7of SGCG. MyoD, a muscle marker, is expressed from the retrovirus (middlepanel) and desmin, a muscle marker, is induced from MyoD (right panel)indicating that these cells are viable models for muscle disease. Theleft hand panel (Hoechst) shows nuclei.

DETAILED DESCRIPTION OF THE INVENTION

Mutations in the gene encoding γ-sarcoglycan, Sgcg, lead to musculardystrophy, a disease with muscle degeneration, failed regeneration andmuscle weakness. One strategy examined herein for the treatment ofgenetic forms of muscular dystrophy is exon skipping. Exclusion ofcertain exon(s) from final transcripts, or exon skipping, can beachieved by blocking essential splice sites using one or more antisensepolynucleotides (AONs). By inducing splicing around mutation-bearingexons, an internally deleted but potentially functional protein isproduced. γ-sarcoglycan is a membrane-associated protein that is part ofthe dystrophin protein complex, a complex that stabilizes the musclemembrane during muscle contraction. The Sgcg gene is composed of 8exons. The most common mutation in Sgcg is the deletion of thymidine 525in exon 6 (525ΔT), causing the production of 19 missense amino acids anda premature stop codon. Skipping exons 4-7 restores the proper proteinreading frame, resulting in an internally truncated γ-sarcoglycanprotein. This truncated form of γ-sarcoglycan reduces the full-lengthprotein from 291 amino acids to 157 amino acids and retains theintracellular region, transmembrane domain and the crucial cysteine-richmotif at the carboxy-terminus.

The γ-sarcoglycan gene is conserved between human, mouse and Drosophila,and both fly and mouse models of γ-sarcoglycan gene mutations havepreviously been generated. The amino acid sequence of human gammasarcoglycan is set forth in SEQ ID NO: 5, while the amino acid sequenceof mouse gamma sarcoglycan is set forth in SEQ ID NO: 6. The truncatedmurine or human γ-sarcoglycan resulting from skipping exon 4-7 has beentermed “mini-Sgcg.” The amino acid sequence of human mini-Sgcg resultingfrom skipping exon 4-7 is set forth in SEQ ID NO: 7, while the aminoacid sequence of mouse mini-Sgcg resulting from skipping exon 4-7 is setforth in SEQ ID NO: 8. The disclosure also contemplates, in variousembodiments, human or mouse mini-Sgcg polypeptides in which one or moreof exon 4, exon 5, exon 6 and exon 7 are deleted in any combination.Thus, mini-Sgcg polypeptides are contemplated in which exon 4-7 arevariously deleted, including but not limited to combinations in which:(i) exon 4 and exon 5 are deleted; (ii) exon 5 and exon 6 are deleted;(iii) exon 4 and exon 6 are deleted; (iv) exon 4 and exon 7 are deleted;(v) exon 5 and exon 7 are deleted; (vi) exon 6 and exon 7 are deleted;(vii) exon 4, exon 5 and exon 6 are deleted; (viii) exon 5, exon 6 andexon 7 are deleted; (ix) exon 4, exon 6 and exon 7 are deleted; (x) exon4 is deleted; (xi) exon 5 is deleted; (xii) exon 6 is deleted; or (xiii)exon 7 is deleted. The disclosure also contemplates correspondingpolynucleotides that encode each of the foregoing mini-Sgcgpolypeptides.

In some aspects, the disclosure provides an isolated mini-gammasarcoglycan polypeptide, wherein the gamma sarcoglycan comprises atleast one deletion in an exon selected from the group consisting of exon4, exon 5, exon 6 and exon 7. In some embodiments, the mini-gammasarcoglycan polypeptide of the disclosure comprises a sequence as setforth in SEQ ID NO: 7 or SEQ ID NO: 8.

In additional aspects, the disclosure provides an isolated polypeptidethat is at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99% or 100% identical to a mini-gamma sarcoglycanpolypeptide of the disclosure. In further aspects, the disclosureprovides an isolated polypeptide that is about 75%, about 80%, about85%, about 90%, about 95% or about 99% identical to a mini-gammasarcoglycan polypeptide of the disclosure. In still further aspects, thedisclosure provides an isolated polypeptide that is at least 90% toabout 100%, or at least 95% to about 100%, or at least about 95% toabout 99% identical to a mini-gamma sarcoglycan polypeptide of thedisclosure.

Data provided herein demonstrates that the mini-Sgcg protein is producedin transgenic Drosophila, where it localizes normally to the plasmamembrane. The structures of the full-length γ-sarcoglycan and thetruncated protein after exon skipping are shown in FIGS. 1 and 2,respectively.

The disclosure thus provides one or more isolated antisensepolynucleotide(s) wherein the one or more polynucleotide(s) specificallyhybridizes to an exon target region of a gamma sarcoglycan RNA, whereinthe exon is selected from the group consisting of exon 4 (SEQ ID NO: 1),exon 5 (SEQ ID NO: 2), exon 6 (SEQ ID NO: 3), exon 7 (SEQ ID NO: 4) anda combination thereof.

As used herein, “hybridization” means an interaction between two orthree strands of nucleic acids by hydrogen bonds in accordance with therules of Watson-Crick DNA complementarity, Hoogstein binding, or othersequence-specific binding known in the art. Hybridization can beperformed under different stringency conditions known in the art.“Specifically hybridize,” as used herein, is hybridization that allowsfor a stabilized duplex between polynucleotide strands that arecomplementary or substantially complementary. For example, apolynucleotide strand having 21 nucleotide units can base pair withanother polynucleotide of 21 nucleotide units, yet only 19 bases on eachstrand are complementary or substantially complementary, such that the“duplex” has 19 base pairs. The remaining bases may, for example, existas 5′ and/or 3′ overhangs. Further, within the duplex, 100%complementarity is not required; substantial complementarity isallowable within a duplex. Substantial complementarity refers to 75% orgreater complementarity. For example, a mismatch in a duplex consistingof 19 base pairs results in 94.7% complementarity, rendering the duplexsubstantially complementary.

It is noted here that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

It is also noted that the term “about” as used herein is understood tomean approximately.

Antisense Polynucleotides/Polynucleotide Design

According to a first aspect of the invention, there is provided anantisense polynucleotide capable of binding to a selected target toinduce exon skipping. To induce exon skipping in exons of the gammasarcoglycan gene transcript, the antisense polynucleotide is selectedbased on the exon sequences shown in Tables 1 and 2. The disclosure alsoprovides a combination or “cocktail” of two or more antisensepolynucleotides capable of binding to a selected target or targets toinduce exon skipping. The exon skipping contemplated herein inducesexclusion of exons 4, 5, 6, and/or 7 so as to generate an in-frame,internally truncated gamma sarcoglycan protein. Excluding exons 4, 5, 6and 7 results in the generation of an internally truncated proteinlacking 135 amino acids, while deleting exon 5 results in an internallydeleted, in-frame protein lacking 40 amino acids. The internallytruncated proteins, termed mini-Sgcg, retains the capacity to interactwith dystrophin and its associated proteins and stabilize cardiac andskeletal muscle cells.

Within the context of the disclosure, preferred target site(s) are thoseinvolved in mRNA splicing (i.e., splice donor sites, splice acceptorsites or exonic splicing enhancer elements). Splicing branch points andexon recognition sequences or splice enhancers are also potential targetsites for modulation of mRNA splicing.

Thus, in various embodiments, one, two three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or moreantisense polynucleotides are used to induce exon skipping of a gammasarcoglycan nucleic acid. The choice of the number of antisensepolynucleotides can be determined empirically by one of ordinary skillin the art. The person of ordinary skill can individually test therelative ability of compositions comprising one, two three, four or moreantisense polynucleotides to produce a protein product of interest invitro. Briefly, and in one specific embodiment, a composition comprisinga single antisense polynucleotide that is designed to specificallyhybridize (i.e., block) a splice acceptor site in exon 4 of a gammasarcoglycan nucleic acid is added to a culture of fibroblasts obtainedfrom a patient harboring a mutation in gamma sarcoglycan. Next, thefibroblasts are induced to adopt a myogenic lineage via forced MyoDexpression (see Example 2 for details), and the resulting myotubes aretested for surface expression of a mini-Sgcg protein via, for exampleand without limitation, an immunofluorescence experiment. Furtherimmunofluorescent analysis of the myotubes can be conducted to identifywhether additional sarcoglycans (i.e., α-, β- and δ-sarcoglycan) areco-localized with mini-Sgcg in myotubes. Such co-localization of themembers of the sarcoglycan complex associated with muscle membranesindicates that the mini-Sgcg that is produced following administrationof the composition comprising a single antisense polynucleotide is ableto effectively induce exon skipping of the gamma sarcoglycan nucleicacid to result in a truncated protein that retained its ability toassociate with the other members of the sarcoglycan complex, as well asembed in a muscle membrane. Similar experiments may be conducted withcompositions that individually comprise two, three, four, five or moreantisense polynucleotides, each designed to specifically hybridize to anexon of a gamma sarcoglycan nucleic acid.

To identify and select antisense polynucleotides suitable for use in themodulation of exon skipping, a nucleic acid sequence whose function isto be modulated must first be identified. This may be, for example, agene (or mRNA transcribed form the gene) whose expression is associatedwith a particular disorder or disease state, or a nucleic acid moleculefrom an infectious agent. Within the context of the disclosure, suitabletarget site(s) are those involved in mRNA splicing (e.g., splice donorsites, splice acceptor sites, or exonic splicing enhancer elements).Splicing branch points and exon recognition sequences or spliceenhancers are also potential target sites for modulation of mRNAsplicing contemplated by the disclosure.

TABLE 2 Table of exon coordinates based on the UCSC Human Genome Build19. exon start exon end exon start + 30 exon end + 30 exon 4 2382476823824856 23824738 23824886 exon 5 23853497 23853617 23853467 23853647exon 6 23869553 23869626 23869523 23869656 exon 7 23894775 23894899 23894725* 23894929 Sgcg exons per UCSC hg19, transcript NM_000231 *−50from exon start because of T rich region

Those of skill in the art can readily design antisense polynucleotidesaccording to the present disclosure. For example, general teachings inthe art include, but are not limited to, Aartsma-Rus et al., Methods MolBiol. 867: 117-29 (2012); Aartsma-Rus et al., Methods Mol Biol. 867:97-116 (2012); van Roon-Mom et al., Methods Mol Biol. 867: 79-96 (2012),each of which is incorporated herein by reference. General guidelinesalso include attempting to avoid 3 consecutive G or C nucleotides,choosing lengths and sequences that favor self structure (hairpinningwill be avoided), and avoiding those sequences likely to form primerdimers. In some embodiments, an antisense polynucleotide of thedisclosure is one that is designed to specifically hybridize to an exonor an intron-exon boundary, such that the antisense polynucleotidespecifically hybridizes to a sequence that is completely within an exonof a gamma sarcoglycan nucleic acid, or about one nucleotide of theantisense polynucleotide spans said intron-exon boundary when theantisense polynucleotide is specifically hybridized to the gammasarcoglycan nucleic acid. In some embodiments wherein the antisensepolynucleotide specifically hybridizes to a sequence that is completelywithin an exon, it is contemplated that a terminus of the antisensepolynucleotide is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morenucleotides from a terminus of the exon. The intron-exon boundary foreach of exons 4, 5, 6, and 7 is shown in Table 1. In furtherembodiments, an antisense polynucleotide of the disclosure is one thatis designed to specifically hybridize to an intron-exon boundary of agamma sarcoglycan nucleic acid, such that about 2, 3, 4, 5, 6, 7, 8, 9,10 or more nucleotides of the antisense polynucleotide span saidintron-exon boundary. It is understood that a nucleotide can “span theintron-exon boundary” on either the exon side or intron side. Thus, anantisense polynucleotide that specifically and predominantly hybridizesto intronic sequence and only hybridizes to one nucleotide of anadjoining exon would “span the intron-exon boundary” by one nucleotide.Similarly, an antisense polynucleotide that specifically hybridizes toexonic sequence and only hybridizes to one nucleotide of an adjoiningintron would “span the intron-exon boundary” by one nucleotide. In anyof the aforementioned embodiments, the antisense polynucleotide is atleast about 10 nucleotides and up to about 15, 20, 25, 30, 35, 40, 45,50 or more nucleotides in length. Lengths of antisense polynucleotidescontemplated by the disclosure are discussed in more detail below.

In some aspects, the disclosure provides pharmaceutical compositionscomprising an antisense polynucleotide to induce exon skipping of agamma sarcoglycan nucleic acid, such that a “mini-Sgcg” protein isproduced that has the ability to (a) effectively associate with othermembers of the sarcoglycan complex (i.e., α-, β- and δ-sarcoglycan) and(b) correctly embed in a muscle membrane. In some embodiments, methodsdescribed herein result in the restoration of a sarcoglycan at a musclemembrane surface, such that about 1% of the gamma sarcoglycan protein isrestored relative to the amount of gamma sarcoglycan protein at a musclemembrane in the absence of administration of the pharmaceuticalcomposition. In further embodiments, methods described herein result inthe restoration of a sarcoglycan protein at the muscle membrane surface,such that about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%,about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%,about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%,about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%,about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold or moreof the gamma sarcoglycan protein is restored relative to the amount ofgamma sarcoglycan protein at the muscle membrane in the absence ofadministration of the pharmaceutical composition. Such restoration ofgamma sarcoglycan protein at the muscle membrane can be determined byone of ordinary skill in the art by, for example and without limitation,obtaining a muscle biopsy from the patient and performingimmunofluorescence with an antibody that has specific binding affinityfor mini-Sgcg protein.

Polynucleotides

Products, uses and methods of the disclosure comprise one or morepolynucleotides. As used herein, a “polynucleotide” is an oligomercomprised of nucleotides. A polynucleotide may be comprised of DNA, RNAmodified forms thereof, or a combination thereof.

The term “nucleotide” or its plural as used herein is interchangeablewith modified forms as discussed herein and otherwise known in the art.In certain instances, the art uses the term “nucleobase” which embracesnaturally occurring nucleotides as well as modifications of nucleotidesthat can be polymerized. Thus, nucleotide or nucleobase means thenaturally occurring nucleobases adenine (A), guanine (G), cytosine (C),thymine (T) and uracil (U) as well as non-naturally occurringnucleobases such as xanthine, diaminopurine, 8-oxo-N6-methyladenine,7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin,N′,N′-ethano-2,6-diaminopurine, 5-methylcytosine (mC),5-(C₃-C₆)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-tr-iazolopyridin, isocytosine,isoguanine, inosine and the “non-naturally occurring” nucleobasesdescribed in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freierand Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp4429-4443. The term “nucleobase” also includes not only the known purineand pyrimidine heterocycles, but also heterocyclic analogues andtautomers thereof. Further naturally and non-naturally occurringnucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan,et al.), in Chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design1991, 6, 585-607, each of which is hereby incorporated by reference inits entirety). In various aspects, polynucleotides also include one ormore “nucleosidic bases” or “base units” which include compounds such asheterocyclic compounds that can serve like nucleobases, includingcertain “universal bases” that are not nucleosidic bases in the mostclassical sense but serve as nucleosidic bases. Universal bases include3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole),and optionally substituted hypoxanthine. Other desirable universal basesinclude pyrrole, and diazole or triazole derivatives, including thoseuniversal bases known in the art.

Polynucleotides may also include modified nucleobases. A “modified base”is understood in the art to be one that can pair with a natural base(e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or canpair with a non-naturally occurring base. Exemplary modified bases aredescribed in EP 1 072 679 and WO 97/12896, the disclosures of which areincorporated herein by reference. Modified nucleobases include, withoutlimitation, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified bases include tricyclic pyrimidinessuch as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases mayalso include those in which the purine or pyrimidine base is replacedwith other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine and 2-pyridone. Additional nucleobases include thosedisclosed in U.S. Pat. No. 3,687,808, those disclosed in The ConciseEncyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed byEnglisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these bases are useful for increasingthe binding affinity of the polynucleotide and include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. and are, in certain aspects, combinedwith 2′-O-methoxyethyl sugar modifications. See, U.S. Pat. No.3,687,808, U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273;5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617;5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,750,692 and 5,681,941, thedisclosures of which are incorporated herein by reference.

Modified polynucleotides are contemplated for use wherein both one ormore sugar and/or one or more internucleotide linkage of the nucleotideunits in the polynucleotide is replaced with “non-naturally occurring”sugars (i.e., sugars other than ribose or deoxyribose) orinternucleotide linkages, respectively. In one aspect, this embodimentcontemplates a peptide nucleic acid (PNA). In PNA compounds, thesugar-backbone of a polynucleotide is replaced with an amide-containing(e.g., peptide bonds between N-(2-aminoethyl)-glycine units) backbone.See, for example U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, andNielsen et al., Science, 1991, 254, 1497-1500, the disclosures of whichare herein incorporated by reference.

Modified polynucleotides may also contain one or more substituted sugargroups. In one aspect, a modification of the sugar includes LockedNucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugargroup. The linkage is in certain aspects a methylene (—CH₂—)_(n) groupbridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2.LNAs and preparation thereof are described in WO 98/39352 and WO99/14226, the disclosures of which are incorporated herein by reference.

To avoid degradation of pre-mRNA during duplex formation with theantisense polynucleotides, the antisense polynucleotides used in themethod may be adapted to minimize or prevent cleavage by endogenousRNase H. This property is advantageous because the treatment of the RNAwith the unmethylated polynucleotides either intracellularly or in crudeextracts that contain RNase H leads to degradation of thepre-mRNA:antisense polynucleotide duplexes. Any form of modifiedantisense polynucleotide that is resistant to such degradation, or doesnot induce such degradation, is contemplated by the disclosure.Non-limiting examples of antisense molecules which, when duplexed withRNA, are not cleaved by cellular RNase H are polynucleotides comprising2′-O-methyl derivatives of nucleotides. 2′-O-methyl-oligoribonucleotidesare very stable in a cellular environment and in animal tissues, andtheir duplexes with RNA have higher T_(m) values than their ribo- ordeoxyribo-counterparts.

Antisense polynucleotides that do not activate RNase H can be made inaccordance with known techniques (see, for example and withoutlimitation, U.S. Pat. No. 5,149,797). Such antisense polynucleotides,which may be deoxyribonucleotide or ribonucleotide sequences, simplycontain any structural modification which sterically hinders or preventsbinding of RNase H to a duplex molecule containing the polynucleotide asone member thereof, which structural modification does not substantiallyhinder or disrupt duplex formation. Because the portions of thepolynucleotide involved in duplex formation are substantially differentfrom those portions involved in RNase H binding thereto, numerousantisense molecules that do not activate RNase H are available.(Activation is used in this sense to refer to RNase H degradation,whether as a result of a substrate not being susceptible to suchdegradation or such substrate failing to induce degradation.) Forexample, such antisense molecules may be polynucleotides wherein atleast one, or all, of the inter-nucleotide bridging phosphate residuesare modified phosphates, such as methyl phosphonates, methylphosphorothioates, phosphoromorpholidates, phosphoropiperazidates and/orphosphoramidates. For example, every other one of the internucleotidebridging phosphate residues may be modified as described. In anothernon-limiting example, such antisense polynucleotides are polynucleotideswherein at least one, or all, of the nucleotides contain a 2′ carbonbound to a lower alkyl moiety (e.g., C₁-C₄, linear or branched,saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl,1-propenyl, 2-propenyl, and isopropyl). For example, every other one ofthe nucleotides may be modified as described.

Methods of making polynucleotides of a predetermined sequence arewell-known. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides andAnalogues, 1st Ed. (Oxford University Press, New York, 1991).Solid-phase synthesis methods are preferred for both polyribonucleotidesand polydeoxyribonucleotides (the well-known methods of synthesizing DNAare also useful for synthesizing RNA). Polyribonucleotides can also beprepared enzymatically. Non-naturally occurring nucleobases can beincorporated into the polynucleotide, as well. See, e.g., U.S. Pat. No.7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J.Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949(1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am.Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc.,124:13684-13685 (2002).

Polynucleotides contemplated herein range from about 5 nucleotides toabout 50 nucleotides in length. In some embodiments, the polynucleotideis between at least 5 nucleotides and at least 20 nucleotides, betweenat least 5 nucleotides and at least 30 nucleotides or between at least 5nucleotides and at least 50 nucleotides.

In further embodiments, a polynucleotide contemplated by the disclosureis about 5 to about 60, 70, 80, 90, 100 or more nucleotides in length,about 5 to about 90 nucleotides in length, about 5 to about 80nucleotides in length, about 5 to about 70 nucleotides in length, about5 to about 60 nucleotides in length, about 5 to about 50 nucleotides inlength about 5 to about 45 nucleotides in length, about 5 to about 40nucleotides in length, about 5 to about 35 nucleotides in length, about5 to about 30 nucleotides in length, about 5 to about 25 nucleotides inlength, about 5 to about 20 nucleotides in length, about 5 to about 15nucleotides in length, about 5 to about 10 nucleotides in length, andall polynucleotides intermediate in length of the sizes specificallydisclosed to the extent that the polynucleotide is able to achieve thedesired result. Accordingly, polynucleotides of 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 ormore nucleotides in length are contemplated.

The polynucleotides of the disclosure are approximately 40% GC to about60% GC, with a Tm of about 48° C. or higher.

Another modification of the polynucleotides of the invention involveschemically linking the polynucleotide to one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the polynucleotide. Such moieties include, but are not limitedto, lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

Therapeutic Agents

The compounds of the disclosure also can be used as a prophylactic ortherapeutic, which may be utilized for the purpose of treatment of agenetic disease.

In one embodiment the disclosure provides antisense polynucleotides thatbind to a selected target in the gamma sarcoglycan pre-mRNA to induceefficient and consistent exon skipping described herein in atherapeutically or prophylactically effective amount admixed with apharmaceutically acceptable carrier, diluent, or excipient.

A pharmaceutically acceptable carrier refers, generally, to materialsthat are suitable for administration to a subject wherein the carrier isnot biologically harmful, or otherwise, causes undesirable effects. Suchcarriers are typically inert ingredients of a medicament. Typically acarrier is administered to a subject along with an active ingredientwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of a pharmaceuticalcomposition in which it is contained. Suitable pharmaceutical carriersare described in Martin, Remington's Pharmaceutical Sciences, 18th Ed.,Mack Publishing Co., Easton, Pa., (1990), incorporated by referenceherein in its entirety.

In a more specific form of the disclosure there are providedpharmaceutical compositions comprising therapeutically effective amountsof an antisense polynucleotide together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions include diluents of various buffer content(e.g., phosphate, Tris-HCl, acetate), pH and ionic strength andadditives such as detergents and solubilizing agents (e.g., Tween 80,Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). The material may beincorporated into particulate preparations of polymeric compounds suchas, for example and without limitation, polylactic acid or polyglycolicacid, or into liposomes. Hylauronic acid may also be used. Suchcompositions may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the disclosedcompositions. The compositions may be prepared in liquid form, or may bein dried powder, such as lyophilized form.

It will be appreciated that pharmaceutical compositions providedaccording to the disclosure may be administered by any means known inthe art. Preferably, the pharmaceutical compositions for administrationare administered by injection, orally, or by the pulmonary, or nasalroute. The antisense polynucleotides are, in various embodiments,delivered by intravenous, intra-arterial, intraperitoneal,intramuscular, or subcutaneous routes of administration.

The antisense molecules of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such pro-drugs, andother bioequivalents.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

For polynucleotides, preferred examples of pharmaceutically acceptablesalts include, but are not limited to, (a) salts formed with cationssuch as sodium, potassium, ammonium, magnesium, calcium, polyamines suchas spermine and spermidine; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid; (c) salts formed withorganic acids such as, for example, acetic acid, oxalic acid, tartaricacid, succinic acid, maleic acid, fumaric acid, gluconic acid, citricacid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmiticacid, alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid; and (d) salts formed from elemental anionssuch as chlorine, bromine, and iodine. The pharmaceutical compositionsof the disclosure may be administered in a number of ways depending uponwhether local or systemic treatment is desired and upon the area to betreated. Administration may be topical (including ophthalmic and tomucous membranes including rectal delivery), pulmonary, e.g., byinhalation of powders or aerosols, (including by nebulizer,intratracheal, intranasal, epidermal and transdermal), oral orparenteral. Parenteral administration includes intravenous,intra-arterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Polynucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

The pharmaceutical formulations of the disclosure, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly bringing intoassociation the active ingredients with liquid carriers or finelydivided solid carriers or both, and then, if necessary, shaping theproduct.

Combination therapy with an additional therapeutic agent is alsocontemplated by the disclosure. Examples of therapeutic agents that maybe delivered concomitantly with a composition of the disclosure include,without limitation, a glucocorticoid steroid (for example and withoutlimitation, prednisone and deflazacort), an angiotensin convertingenzyme inhibitor, a beta adrenergic receptor blocker, an anti-fibroticagent and a combination thereof.

Gene Therapy

In some aspects, the disclosure provides methods of expressing amini-gamma sarcoglycan in a cell. In any of the aspects or embodimentsof the disclosure, the cell is a mammalian cell. In any of the aspectsor embodiments of the disclosure, the cell is in a human and the humanis in need of the mini-gamma sarcoglycan. Accordingly, in some aspectsthe disclosure provides gene therapy methods for expressing a mini-gammasarcoglycan in a cell.

In some embodiments, a vector (e.g., an expression vector) comprising apolynucleotide of the invention to direct expression of thepolynucleotide in a suitable host cell. Such vectors are useful, e.g.,for amplifying the polynucleotides in host cells to create usefulquantities thereof, and for expressing proteins using recombinanttechniques. In some embodiments, the vector is an expression vectorwherein a polynucleotide of the invention is operatively linked to apolynucleotide comprising an expression control sequence. Autonomouslyreplicating recombinant expression constructs such as plasmid and viralDNA vectors incorporating polynucleotides of the disclosure arespecifically contemplated. Expression control DNA sequences includepromoters, enhancers, and operators, and are generally selected based onthe expression systems in which the expression construct is to beutilized. In some embodiments, promoter and enhancer sequences areselected for the ability to increase gene expression, while operatorsequences may be selected for the ability to regulate gene expression.Expression constructs of the invention may also include sequencesencoding one or more selectable markers that permit identification ofhost cells bearing the construct. Expression constructs may also includesequences that facilitate, and preferably promote, homologousrecombination in a host cell. Expression constructs of the disclosurealso include, in various embodiments, sequences necessary forreplication in a host cell.

Exemplary expression control sequences include promoter/enhancersequences, e.g., cytomegalovirus promoter/enhancer [Lehner et al., J.Clin. Microbiol., 29: 2494-2502, 1991; Boshart et al., Cell, 41:521-530, (1985)]; Rous sarcoma virus promoter [Davis et al., Hum. GeneTher., 4: 151, (1993)]; and simian virus 40 promoter, for expression ina target mammalian cell, the promoter being operatively linked upstream(i.e., 5′) of the polypeptide coding sequence (the disclosures of thecited references are incorporated herein by reference in their entiretyand particularly with respect to the discussion of expression controlsequences). In another variation, the promoter is a muscle-specificpromoter. The polynucleotides of the invention may also optionallyinclude a suitable polyadenylation sequence (e.g., the SV40 or humangrowth hormone gene polyadenylation sequence) operably linked downstream(i.e., 3′) of the polypeptide coding sequence.

If desired, a polynucleotide of the disclosure also optionally comprisesa nucleotide sequence encoding a secretory signal peptide fused in framewith the polypeptide sequence. The secretory signal peptide directssecretion of the polypeptide of the invention by the cells that expressthe polynucleotide, and is cleaved by the cell from the secretedpolypeptide. The polynucleotide may further optionally comprisesequences whose only intended function is to facilitate large scaleproduction of the vector, e.g., in bacteria, such as a bacterial originof replication and a sequence encoding a selectable marker. However, ifthe vector is administered to an animal, such extraneous sequences arepreferably at least partially cleaved. One can manufacture andadminister polynucleotides for gene therapy using procedures that havebeen described in the literature for other transgenes. See, e.g., Isneret al., Circulation, 91: 2687-2692, 1995; Isner et al., Human GeneTherapy, 7: 989-1011, 1996; Wang et al., Mol Ther. 20(8):1501-7 (2012);and Zhang et al., Hum Mol Genet. 22(18): 3720-9 (2013); each of which isincorporated herein by reference in its entirety.

In some embodiments, a “naked” transgene encoding a mini-gammasarcoglycan described herein (i.e., a transgene without a viral,liposomal, or other vector to facilitate transfection) is employed.

Vectors also are useful for “gene therapy” treatment regimens, wherein,for example, a polynucleotide encoding a mini-gamma sarcoglycan isintroduced into a subject suffering from or at risk of suffering from amuscular dystrophy in a form that causes cells in the subject to expressthe mini-gamma sarcoglycan in vivo. Any suitable vector may be used tointroduce a polynucleotide that encodes a mini-gamma sarcoglycan intothe host. Exemplary vectors that have been described in the literatureinclude replication deficient retroviral vectors, including but notlimited to lentivirus vectors [Kim et al., J. Virol., 72(1): 811-816,(1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46];parvoviral vectors, such as adeno-associated viral (AAV) vectors [U.S.Pat. No. 5,474,9351; 5,139,941; 5,622,856; 5,658,776; 5,773,289;5,789,390; 5,834,441; 5,863,541; 5,851,521; 5,252,479; Gnatenko et al.,J. Invest. Med., 45: 87-98, (1997)]; adenoviral (AV) vectors [U.S. Pat.Nos. 5,792,453; 5,824,544; 5,707,618; 5,693,509; 5,670,488; 5,585,362;Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584, (1992);Stratford Perricaudet et al., J. Clin. Invest., 90: 626-630, (1992); andRosenfeld et al., Cell, 68: 143-155, (1992)]; an adenoviraladeno-associated viral chimeric [U.S. Pat. No. 5,856,152] or a vacciniaviral or a herpesviral vector [U.S. Pat. Nos. 5,879,934; 5,849,571;5,830,727; 5,661,033; 5,328,688]; Lipofectin mediated gene transfer(BRL); liposomal vectors [U.S. Pat. No. 5,631,237]; and combinationsthereof. Additionally contemplated by the disclosure for introducing apolynucleotide encoding a mini-gamma sarcoglycan into a subject is aplasmid vector [see, e.g., Dean, Am J Physiol Cell Physiol. 289(2):C233-45 (2005); Kaufman et al., Gene Ther. 17(9): 1098-104 (2010);Magnusson et al., J Gene Med. 13(7-8): 382-91 (2011)]. For example andwithout limitation, any pBR- or pUC-derived plasmid vector iscontemplated. All of the foregoing documents are incorporated herein byreference in their entirety and particularly with respect to theirdiscussion of expression vectors. Any of these expression vectors can beprepared using standard recombinant DNA techniques described in, e.g.,Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubelet al., Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons, New York, N.Y. (1994). Optionally, theviral vector is rendered replication-deficient by, e.g., deleting ordisrupting select genes required for viral replication.

Other non-viral delivery mechanisms contemplated include calciumphosphate precipitation [Graham and Van Der Eb, Virology, 52: 456-467,1973; Chen and Okayama, Mol. Cell Biol., 7: 2745-2752, (1987); Rippe etal., Mol. Cell Biol., 10: 689-695, (1990)], DEAE-dextran [Gopal, Mol.Cell Biol., 5: 1188-1190, (1985)], electroporation [Tur-Kaspa et al.,Mol. Cell Biol., 6: 716-718, (1986); Potter et al., Proc. Nat. Acad.Sci. USA, 81: 7161-7165, (1984)], direct microinjection [Harland andWeintraub, J. Cell Biol., 101: 1094-1099, (1985)], DNA-loaded liposomes[Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190, (1982); Fraleyet al., Proc. Natl. Acad. Sci. USA, 76: 3348-3352, (1979); Felgner, SciAm., 276(6): 102-6, (1997); Felgner, Hum Gene Ther., 7(15): 1791-3,(1996)], cell sonication [Fechheimer et al., Proc. Natl. Acad. Sci. USA,84: 8463-8467, (1987)], gene bombardment using high velocitymicroprojectiles [Yang et al., Proc. Natl. Acad. Sci USA, 87: 9568-9572,(1990)], and receptor-mediated transfection [Wu and Wu, J. Biol. Chem.,262: 4429-4432, (1987); Wu and Wu, Biochemistry, 27: 887-892, (1988); Wuand Wu, Adv. Drug Delivery Rev., 12: 159-167, (1993)].

The expression vector (or the mini-gamma sarcoglycan discussed herein)may be entrapped in a liposome.

In some embodiments, transferring a naked DNA expression construct intocells is accomplished using particle bombardment, which depends on theability to accelerate DNA coated microprojectiles to a high velocityallowing them to pierce cell membranes and enter cells without killingthem [Klein et al., Nature, 327: 70-73, (1987)]. Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force [Yang et al., Proc. Natl. Acad. Sci USA,87: 9568-9572, (1990)]. The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold beads.

In embodiments employing a viral vector, preferred polynucleotides stillinclude a suitable promoter and polyadenylation sequence as describedabove. Moreover, it will be readily apparent that, in these embodiments,the polynucleotide further includes vector polynucleotide sequences(e.g., adenoviral polynucleotide sequences) operably connected to thesequence encoding a polypeptide of the disclosure.

The disclosure further provides a cell that comprises the polynucleotideor the vector, e.g., the cell is transformed or transfected with apolynucleotide encoding a mini-gamma sarcoglycan of the disclosure orthe cell is transformed or transfected with a vector comprising apolynucleotide encoding the mini-gamma sarcoglycan.

Polynucleotides of the disclosure may be introduced into the host cellas part of a circular plasmid, or as linear DNA comprising an isolatedprotein coding region or a viral vector. Methods for introducing DNAinto the host cell, which are well known and routinely practiced in theart, include transformation, transfection, electroporation, nuclearinjection, or fusion with carriers such as liposomes, micelles, ghostcells, and protoplasts. As stated above, such host cells are useful foramplifying the polynucleotides and also for expressing the polypeptidesof the invention encoded by the polynucleotide. The host cell may beisolated and/or purified. The host cell also may be a cell transformedin vivo to cause transient or permanent expression of the polypeptide invivo. The host cell may also be an isolated cell transformed ex vivo andintroduced post-transformation, e.g., to produce the polypeptide in vivofor therapeutic purposes.

Kits

The disclosure also provides kits for treatment of a patient with agenetic disease such as LGMD2C. In one aspect, the kit comprises anantisense polynucleotide as disclosed herein, optionally in a container,and a package insert, package label, instructions or other labeling.

In a further embodiment, a kit is provided that comprises an additionalpolynucleotide, wherein the additional polynucleotide specificallyhybridizes to an exon in a gamma sarcoglycan RNA.

Those of ordinary skill in the art will appreciate that applications ofthe above method has wide application for identifying antisensemolecules suitable for use in the treatment of many other diseases.

EXAMPLES Example 1 Rescue Efficiency of Mammalian Full Lengthγ-Sarcoglycan and Mini-Sgcg in Sarcoglycan Null Flies

The sarcoglycans are conserved between Drosophila and mammals.γ-sarcoglycan null flies develop symptoms similar to mammals. Transgenicflies expressing full-length murine γ-sarcoglycan and mini-Sgcg, weregenerated and it was found that mini-Sgcg protein correctly localizes atthe plasma membrane of fly muscle cells.

γ/δ-sarcoglycan null flies (Sgcd⁸⁴⁰) were previously generated andcharacterized [Allikian et al., Hum Mol Genet 16: 2933-2943 (2007)].Using PCR and Southern blot, it has been shown that exons 1 to 3 andpartial exon 4 out of the 6 exons in the Drosophila Sgcd gene is deletedin Sgcd⁸⁴⁰ flies.

To determine whether mini-Sgcg retains the function of the full-lengthprotein, the UAS-GAL4 system was utilized [Brand et al., Development.118: 401-15 (1993)] to express two different sarcoglycan constructs.GAL4 is a transcription factor that recognizes a specific enhancersequence called UAS.

The first construct expresses the full coding sequence of murine Sgcggene from exon 2 to exon 8, referred to as UAS-Sgcg. The secondconstruct comprises the sequence of an Xpress epitope tag and exon 2,exon 3 and exon 8 of murine Sgcg gene only, referred to as UAS-mini-Sgcg(FIG. 1). The shorter construct retains the coding sequence of an intactintracellular domain, transmembrane domain and part of the extracellulardomain including the essential carboxyl-terminus (FIG. 2). A comparisonof the constructs used in Drosophila and mouse is depicted in FIG. 3.One UAS-Sgcg and one UAS-mini-Sgcg transgenic line were generated inmatched genetic backgrounds. A Mef2-GAL4 stock was also obtained. Mef2is a muscle specific driver that promotes GAL4 expression in both heartand skeletal muscle tissue. Flies carrying both Mef2-GAL4 and UAS-Sgcgor UAS-mini-Sgcg produce Sgcg or mini-Sgcg protein specifically in theheart tube and in muscle tissue. To express the full-length andmini-Sgcg in mutant flies, Sgcd⁸⁴⁰, Mef2-GAL4 virgin females werecrossed to either UAS-Sgcg/+ or UAS-mini-Sgcg/+ male flies. Since theSgcd gene is located on X chromosome, all the male progeny from eachcross were null for Sgcd, with half carrying both GAL4 and UAStransgenes. The other half served as an internal negative control. Thiscomparison minimizes the effects of environment and genetic backgroundon behavior assays. Wild-type flies with matched age and geneticbackground served as the positive control.

The data showed that mini-Sgcg has distinct plasma membrane localizationin Sgcd⁸⁴⁰ fly heart tubes (FIG. 4) and in body muscle (FIG. 5). Thispattern is similar to full length Sgcg (FIG. 5). Studies in humanmuscle, mice, and cell expression systems suggest that sarcoglycancomplex assembly is required for the shuttling of γ-sarcoglycan to theplasma membrane [Allikian et al., Traffic 8: 177-83 (2007); Chen et al.,Exp Cell Res. 312: 1610-25 (2006); Crosbie et al., Hum Mol Genet.9:2019-27 (2000)]. Thus the correct subcellular localization of bothproteins implies interaction with the fly sarcoglycan subunits,highlighting the conservation between murine and fly γ-sarcoglycan. Moreimportantly, the results indicated that mini-Sgcg protein retains thefunction of interacting with other components of the dystrophin complex.

Skeletal Muscle Structure and Function in Transgenically Rescued Flies

Patients with LGMD2C display distinct muscle histology from healthyindividuals, including loss of mature muscle fibers, abnormal depositionof fibrotic or fatty tissue and immune cells infiltration [Dubowitz,Muscle disorders in childhood. Saunders, Philadelphia. xiii, 282(1978)]. Sgcd⁸⁴⁰ flies show increased detachment of flight muscle fromexoskeleton, and this finding is more prominent if flies are allowed toexercise [Goldstein et al., Hum Mol Genet. 20: 894-904 (2011)]. Toencourage muscle usage, flies are kept in a 20×20×20 cm box instead ofstandard vials so that they can fly at ease. Flies are aged to 28 daysbefore harvesting for histological examination. Specifically, thoracesare collected, sectioned and stain with hematoxylin and eosin (H&E)using the Carnoy fixation protocol. The frequency of flight musclefracture between mutant flies, rescued flies and wild-type flies arethen compared.

Similar to human patients, Sgcd⁸⁴⁰ flies develop impaired locomotiveability over time [Allikian et al., Hum Mol Genet 16: 2933-43 (2007)].Motility defects are measured using a negative geotaxis assay. Differentfrom most apparatus for bulk measurement, a more “individualized”apparatus was designed that can provide the walking ability measurementof an individual fly as precise as 0.5 cm [Goldstein et al., Hum MolGenet. 20: 894-904 (2011)]. The apparatus is composed of 16 verticalplastic tubes that are 13 cm long with rulers on each side. Briefly, anindividual fly is placed in each tube after anesthesia, allowed torecover for 30 minutes and then tested. To test walking, the flies arethen tapped to the bottom and allowed to climb up for 5 seconds. Thedistance that each fly travels at the end of the 5 seconds is scored.Six trials with a 1-minute interval are performed. Analysis of variance(ANOVA) with a post Tukey test is employed for data analysis in PRISMsoftware.

Assess Heart Function Rescue Using Optical Coherence Tomography (OCT)

Muscular dystrophy patients develop dilated cardiomyopathy due to theimpaired contractility of heart muscle cells. Sgcd⁸⁴⁰ flies also showcardiac malfunction as they age, indicated by enlarged heart tube andreduced fraction shortening [Allikian et al., Hum Mol Genet 16: 2933-43(2007); Goldstein et al., Hum Mol Genet. 20: 894-904 (2011)]. The heartfunction of flies is examined by OCT [Wolf et al., Proc Natl Acad SciUSA. 103: 1394-9 (2006)]. OCT serves as the fly counterpart to theechocardiography (ECHO) used with mammals. The major difference betweenOCT and ECHO is that OCT measures backscattered light instead of sound.OCT determines the end systolic diameter (ESD), end diastolic diameter(EDD), fractional shortening (FS) and heart rate. Twenty to forty fliesfrom each genotype were tested at 7-10 days of age. Data showed thatmini-Sgcg expression reduced the abnormal heart tube dilation in Sgcd⁸⁴⁰flies (FIG. 6), indicating that the mini-Sgcg was functional. Flies willbe assessed at older ages, when the cardiomyopathy is more prevalent.Importantly, the results will be compared to transgenic flies expressingfull-length murine γ-sarcoglycan to determine if the degree ofcorrection is similar between the mini-Sgcg and the full-length Sgcg.

It is expected that expression of either mini-Sgcg or full-length Sgcgwill lead to rescue of disease progression in mutant flies.Specifically, it is expected that less muscle disruption, improvedwalking ability and restored heart function will be seen in mutant flieswith the mini-Sgcg or Sgcg. It is possible that the preparationprocedures of the muscle tissue might interfere with identification ofmuscle tears. Muscle damage induces TGFβ signaling surrounding theinjury sites, which can be visualized by dad-lacZ reporter activity[Goldstein et al., Hum Mol Genet. 20: 894-904 (2011)]. The dad-lacZreporter construct is expected to provide better visualization of muscletearing [Goldstein et al., Hum Mol Genet. 20: 894-904 (2011)]. If thewalking assay is not sensitive enough to detect the improvement broughtby expression of mini-Sgcg, an alternative motility assay that enablesthe examination of a larger number of flies at one time will beperformed [Shcherbata et al., EMBO J 26: 481-93 (2007)]. Mutant flieshave reduced life span [Allikian et al., Hum Mol Genet 16: 2933-43(2007)]. Thus, the lifespan of mini-Sgcg-rescued flies is determined,providing additional evidence of the benefits of mini-Sgcg expression,and the increased lifespan will be quantitated. Data has shown thatexpression of mini-Sgcg significantly improved heart function in Sgcd⁸⁴⁰flies, leading to the expectation that mini-Sgcg or full-length Sgcgwill yield beneficial effects in preventing or treating dystrophicdisease such as LGMD (e.g., LGMD2C). As a comparison, flies that expressDrosophila Sgcd have also been generated for comparison purposes.

Example 2 Mini-Sgcg can Replace Full-Length Sgcg in the γ-SarcoglycanMutant Mouse Model

By characterizing the correction of mutant phenotype in an establishedmouse model, a more accurate prediction of the effect of replacing thefull-length γ-sarcoglycan with the truncated γ-sarcoglycan in humanpatients can be determined.

Transgenic Mouse Expressing Mini-Sgcg in Muscle Using the Human DesminPromoter with the Mini-Sgcg Coding Sequence

To test the function of mini-Sgcg in mice, transgenic mice expressingmurine mini-Sgcg were generated using the desmin promoter whichexpresses in both heart and muscle cells [Pacak et al., Genet VaccinesTher 6: 13 (2008)]. γ-sarcoglycan is required for proper function ofboth heart and skeletal muscle [Zhu et al., FASEB J 16: 1096-1098(2002)]. To assess the rescue efficiency of γ-sarcoglycan sub-domains inboth muscle tissues, the human desmin promoter is used. Desmin is anintermediate filament that is expressed in all muscle tissue, includingheart and skeletal muscle [Su et al., Proc Natl Acad Sci USA 101:6062-6067 (2004)]. Besides being tissue specific, the desmin gene alsobecomes activated during muscle differentiation at around the same timeas the sarcoglycans [Li et al., J Cell Biol 124: 827-841 (1994); Noguchiet al., Eur J Biochem 267: 640-648 (2000)]. Desmin expression levelremains low in dividing myoblasts and reaches a persistently high levelin terminally differentiated myofibers. Minimal sequences of theregulatory region of the human desmin gene have been cloned and havebeen shown to promote high-level target gene expression specifically inboth heart and skeletal muscle [Pacak et al., Genet Vaccines Ther 6: 13(2008)].

Using a CMV-mini-Sgcg construct in a pcDNA™ 3.0 vector (Invitrogen), theCMV promoter was replaced with the desmin promoter. An Xpress epitopetag was inserted at the N-terminus of mini-Sgcg. The purifieddesmin-mini-Sgcg construct (see FIG. 3B) was injected, and atransgene-positive male founder (FIG. 7) was generated. The male founderis being bred to Sgcg^(−/−) mice. Transgene injections continued andfive additional founder lines were generated. To test the function ofthe desmin promoter, C2C12 cells (a cultured muscle cell line) weretransfected with the desmin-mini-Sgcg construct, and it was found thatmini-Sgcg was produced in differentiated myotubes (FIG. 8).

Level of Expression and Subcellular Localization of Mini-Sgcg Using theEpitope Tag

Overexpression of γ-sarcoglycan has been shown to cause severe musculardystrophy in wild-type mice when expression levels are approximately20-fold over normal levels [Zhu et al., FASEB J 16: 1096-8 (2002)]. Thisresult was likely due to the formation of abnormal cytoplasmic proteinaggregates that interfere with sarcoglycan complex assembly and membranetargeting. The copy number of the currently generated single transgeneline appears low, so problems resulting from overexpression are notanticipated. Muscle from desmin-mini-Sgcg transgenic mice are examinedfor normal membrane localization using the Xpress tag. Experiments havebeen conducted with this same construct in C2C12 cells, a muscle cellline. In these cells, mini-Sgcg showed similar localization toendogenous γ-sarcoglycan protein.

To further determine the level of mini-Sgcg production, both heart andskeletal muscle tissue from various transgenic lines are collected andprocessed for immunoblotting experiments. The Xpress epitope tag allowsthe mini-Sgcg protein to be easily detected on the immunoblot throughthe use of an antibody against Xpress. Full-length γ-sarcoglycan isdetected using NCL-g-sarc antibody (Novocastra). Because this monoclonalantibody is raised against a 12-amino-acid peptide within exon 6, onlyfull-length but not mini-Sgcg protein will be detected. Polyclonalantibodies have also been generated that will recognize both full-lengthand mini-Sgcg. Following generation of additional transgenic lines, aline is chosen that shows an expression level near wild-type.

Rescue of γ-Sarcoglycan Mutants by Crossing the Transgene into Sgcg Mice

To determine if mini-Sgcg can replace full-length Sgcg in mammals,mini-Sgcg Tg⁺ (transgene positive) mice are bred to Sgcg^(−/−) mice todetermine whether heart and muscle dysfunction in Sgcg^(−/−) can berescued. To prevent phenotypic drift, Sgcg^(−/−) mice are kept asheterozygotes to reduce selection of spontaneously developed modifiers.To introduce the mini-Sgcg transgene into Sgcg^(−/−) mice, Sgcg^(−/+)are bred to mini-Sgcg Tg⁺ mice. The Tg⁺ Sgcg^(−/+) F1 males are bred tofemales. In F2, Tg⁺ Sgcg^(−/−) mice are produced. Among the littermates,Tg⁺ wild-type mice are used as positive controls while Tg⁻ Sgcg^(−/−)mice are used as negative controls. To measure whether mini-Sgcg canimprove the impaired muscle function in Sgcg^(−/−) mice, the followingaspects are compared between cohorts: subcellular localization ofsarcoglycan proteins, direct interaction between mini-Sgcg and othersarcoglycans, plasma membrane permeability of muscle cells, fibrotictissue deposition (fibrosis) and skeletal muscle function and heartfunction. Histopathology is examined from transgene-rescued mice tocompare to Sgcg^(−/−) and wild-type mice. Muscles are examined forvariation in fiber size, central nucleation and replacement by fibrosisand fat. Sgcg^(−/−) and transgenic mice are all on the C57B16/Jbackground.

Subcellular Localization

In heart and muscle cells lacking γ-sarcoglycan, α-, β-, andδ-sarcoglycans are also greatly reduced from muscle membrane while theirmRNA levels remain normal, indicating that the presence of γ-sarcoglycanis required for the stable membrane localization of other sarcoglycansin the complex [Hack et al., J Cell Sci. 113(14): 2535-44 (2000)]. Totest if the mini-Sgcg protein can restore the proper localization ofother sarcoglycans in the absence of full-length γ-sarcoglycan, sectionsof frozen muscle tissue from Tg⁺ Sgcg^(−/−) animals are examined andimmunofluorescence microscopy is performed using antibodies against α-,β-, and δ-sarcoglycan, respectively. All antibodies are availablecommercially or are those that were previously generated.

Interaction

In wild-type muscle cells, α-, β-, γ- and δ-sarcoglycans form a tightcomplex that localizes at the plasma membrane. γ-sarcoglycan can beco-immunoprecipitated (co-IP) with β-sarcoglycan from muscle tissue[Hack et al., J Cell Sci. 113(14): 2535-44 (2000)]. To examine ifmini-Sgcg can also interact with β-sarcoglycan, co-IP is performed onprotein preparations collected from Tg⁺ Sgcg^(−/−) mice. The muscle isfractionated to isolate the membranes from the myofibrillar components.These microsomal preparations are performed on whole muscle and only themicrosomal fraction is used in the co-IP.

Membrane Permeability

In Sgcg^(−/−) animals, the muscle plasma membrane is weakened andbecomes more permeable to large protein molecules. Evans Blue Dye (EBD)is a small molecule dye that binds tightly to albumin and measuressarcolemmal permeability [Matsuda et al., J Biochem 118: 959-64 (1995)].Mice are injected with EBD and sacrificed 40 hours after injection. EBDis measured by incubating tissues in 1 milliliter of formamide at 55° C.for 2.5 hours and determining the absorbance of the resulting elution at620 nm. Serum creatine kinase (CK) level is also measured using theEnzyChrom™ Creatine Kinase Assay Kit (BioAssay Systems).

Fibrosis

Collagen is the main component of the excessive fibrous tissue. Toquantify collagen deposition, hydroxyproline assays (HOP) are performedto quantitate collagen content. Hydroxyproline is a modified amino acidthat comprises a major portion of collagen. Heart and muscle tissues arecollected and HOP assays are performed according to described methods[Heydemann et al., Neuromuscul Disord 15: 601-9 (2005)], incorporatedherein by reference.

Heart Function

Heart dysfunction is a major direct cause of disability and death inmuscular dystrophy patients. Mice lacking γ-sarcoglycans also developdilated cardiomyopathy. To investigate heart function, echocardiography(ECHO) is performed to measure end-diastolic dimension (EDD),end-systolic dimension (ESD) and fractional shortening (FS).

Analysis of Mice

Mice are analyzed at 12 and 24 weeks of age since these time pointsdisplay both muscle and heart disease. The numbers used will reflect thephysiological studies being conducted and typically require cohorts ofbetween 5 and 10 to show significance (t-test). Additional animals arealso used to provide a supply of tissue for microscopy and Westernblotting.

Strain Ages Number Purpose C57B16/J 12 & 24 wks 10-20 Tissue, echo,histo, IF Des-mini-Sgcg Tg 12 & 24 wks 10-20 Tissue, echo, histo, IFSgcg^(−/−) 12 & 24 wks 10-20 Tissue, echo, histo, IF Des-mini-SgcgTg/Sgcg 12 & 24 wks 10-20 Tissue, echo, histo, IF

Mice are being used because they provide a good model of muscle andheart disease that reflects what is seen in humans with similar genemutations. Over 500 mice of this genotype (Sgcg) have been examined andquantitative methods of phenotyping have been established [Heydemann etal., Neuromuscul Disord 15(9-10): 601-9 (2005); Heydemann et al., J.Clin. Invest 119(12): 3703-12 (2009); Swaggart et al., Physiol Genomics43(1): 24-31 (2011)]. Given the difference in phenotype expected fromthe Drosophila studies, it is anticipated that cohorts of 5-10 mice willbe sufficient.

Expected Results

Several lines of mice harboring des-mini-Sgcg transgenes that expressmini-Sgcg protein at different levels in muscle tissue are expected,with some lines at near-endogenous γ-sarcoglycan levels. It is expectedthat mini-Sgcg is undetectable in other tissues. It is also expectedthat mini-Sgcg protein is enriched at the plasma membrane in wild-typemice. It is possible that some cytoplasmic or perinuclear staining ofmini-Sgcg is observed because the presence of full-length Sgcg maycompete with mini-Sgcg for inclusion in the sarcoglycan complex. Moredistinct plasma membrane staining of mini-Sgcg in Sgcg^(−/−) mice isexpected. A similar pattern of expression has been seen in studies inDrosophila. In Tg⁺ Sgcg^(−/−) muscle, it is expected that mini-Sgcgexpression will restore the membrane localization of other sarcoglycans.Mini-Sgcg is also expected to be present among the proteins associatedwith, and pulled down by, β-sarcoglycan. Improved histopathology,reduced EBD uptake, decreased CK level, less HOP and improved heartfunction are expected in Tg⁺ Sgcg^(−/−) compared to Tg⁻ Sgcg^(−/−)littermates without the transgene. These would all represent animprovement in muscle and heart disease, establishing that mini-Sgcgrescues the Sgcg mutation, as expected.

Based on the Drosophila studies described herein, mini-Sgcg is expectedto have many of the functions of Sgcg. The transgenic mice will alsoallow for the investigation of the interaction with other importantcomponents of the dystrophin complex, such as dystrophin, sarcospan, andinteractions with other transmembrane components.

For exon skipping, fibroblasts have been obtained from human LGMD2Cpatients. A forced MyoD expression approach, described below, is used toinduce these cells into a myogenic lineage [Kimura et al., Hum Mol Genet17: 2507-17 (2008)]. These cells will provide a cell-based environmentin which to test human Sgcg exon skipping.

Example 3 Exon Skipping in Muscle Culture Derived from Human Patients

The rationale for the experiments described below is that exon skippingrequires optimization of antisense polynucleotides and proof-of-functionin vitro.

Use MyoD Transformation to Induce Cultured Primary Human Fibroblasts toBecome Myoblasts

Fibroblasts isolated from two LGMD2C patients carrying a deletion ofexon 6 in the Sgcg gene have been obtained. MyoD is a master regulatorof the muscle differentiation program. Forced expression of MyoD infibroblasts can convert the fibroblasts to a muscle lineage [Lattanzi etal., J Clin Invest 101: 2119-2128 (1998)].

MyoD is a key initiator of the skeletal muscle differentiation program[Weintraub et al., Science 251: 761-766 (1991)]. MyoD is responsible foractivating other essential muscle regulators, such as myocyte enhancerfactor-2 (MEF2) and myogenin. It has been shown that forced expressionof MyoD in fibroblasts is able to convert fibroblasts to myoblasts bothin vitro and in vivo [Kimura et al., Hum Mol Genet 17: 2507-2517(2008)]. Therefore, introducing MyoD into fibroblasts is sufficient toconvert a fibroblast down a myoblast lineage. Once established,myoblasts under the proper conditions can be induced to differentiatefurther into myotubes. This process is applied to human fibroblasts and“MyoD forced fibroblasts” are useful for diagnosing human muscledisease. Myoblasts derived from human patients bearing the γ-sarcoglycanmutation described herein are required in order to test the efficiencyof exon skipping induced by different potential AONs. Using dermalfibroblasts from LGMD2C patients avoids the need for painful musclebiopsies required to obtain myoblasts.

Kimura et al. made a tamoxifen-inducible MyoD construct and inserted theMyoD gene into fibroblast genomes via lentiviral vector. They found thatthe transfected fibroblasts were able to form myotubes both in vivo andin vitro upon administration of tamoxifen. The MyoD vector is used totransfect the fibroblasts obtained from the LGMD2C patients. Thetransfected fibroblasts are expanded without tamoxifen induction andfrozen in small aliquots for future use.

MyoD Forced Fibroblasts Treated with Antisense Polynucleotides InduceExon Skipping of Exons 4-7 of Sgcg

To target specific exons, antisense polynucleotides (AONs) are designedto block splice donor, splice acceptor or exonic splicing enhancer (ESE)sites. Splice donor and splice acceptor sites localize at exon-intronboundaries and have highly conserved sequences. Based on nucleotidesequence and secondary structure of RNA transcripts, ESE sites arepredicted at high accuracy by software such as ESEfinder, which predictsbinding sites for the four most abundant serine/arginine-rich proteinsinvolved in splicing regulation (SR proteins). A series of AONs aredesigned based on the prediction of available software programs. Theefficiency and specificity of different AONs in myotubes converted frompatient fibroblasts is then examined, following a protocol that has beenused to test exon skipping efficiency in primary human myotubes[Aartsma-Rus et al., Hum Mol Genet 12: 907-914 (2003)]. Specifically,fibroblasts are treated with tamoxifen for 24 hours to induce MyoDexpression before switching the fibroblasts to differentiation mediumfor induction of myotube formation. Following 7-14 days of serumdeprivation, the myotubes are transfected with AON usingpolyethylenimine (PEI) for 3 hours in low-serum medium. At 24 hourspost-transfection, total RNA is extracted from the myotube cultures. Theratio of the shorter mRNA transcript composed of only exon 1, 2, 3 and 8to the full-length transcripts is quantified by performing reversetranscriptase-polymerase chain reaction (RT-PCR). The PCR product isfractionated on an agarose gel and the ratio of short/long products iscalculated using Photoshop software. The AONs with highest exon skippingefficiency are selected.

Use of Immunofluorescence (IF) Microscopy to Test Whether theCarboxy-Terminus of γ-Sarcoglycan is Present after Anti-Sense Treatment,and Whether Other Sarcoglycans are Stabilized Using this Approach

Frame shift mutations in the Sgcg gene in patients result in aprematurely terminated protein without a C-terminus. Previous study hasalso shown that the protein product of the Sgcg gene with a 525ΔTmutation was not detected by a rabbit polyclonal antibody raised againthe entire Sgcg protein [McNally et al., Am J Hum Genet 59: 1040-1047(1996)]. This result suggested that the mutant Sgcg gene was not able toproduce a stable protein product. This is also likely to be the case inthe LGMD2C patients from whom the fibroblasts were obtained.

Restoration of the reading frame results in translation of an internallytruncated protein with a normal C-terminus. To visualize the productionof AON-induced protein, IF microscopy is performed using the polyclonalantibody against the full-length γ-sarcoglycan protein on myotubes withand without AON treatment. By using IF, it can also be determinedwhether the smaller γ-sarcoglycan protein localizes to the membrane. IFis also performed to detect other sarcoglycans (e.g., α-, β- andδ-sarcoglycan) and assess whether they have been restored to themembrane in Tg⁺ Sgcg^(−/−) mice.

Expected Results

It is expected that fibroblasts are converted to myoblast-like cellsthat are capable of forming myotubes upon tamoxifen administration.Alternatively, myoblasts are obtained from human patients. It isexpected that several AON treatments will convert a substantial fractionof full-length Sgcg transcript to smaller, internally truncated protein.It is expected that no Sgcg staining is detected by the polyclonalantibody in patient myotubes without AON treatment, while the internallytruncated γ-sarcoglycan protein is expected to be localized at themembrane of a substantial percentage of treated myotubes. Further, it isexpected that other components of the dystrophin complex are restored atthe plasma membrane of the myotubes that show positive γ-sarcoglycanmembrane staining. In the event that the polyclonal antibody is able todetect residual product of the 525ΔT Sgcg gene, an antibody against theC-terminus of the γ-sarcoglycan protein is raised to specifically detectthe truncated γ-sarcoglycan protein.

Example 4 Walking Behavior in Sgcd Null Flies

This experiment was designed to test whether loss of Sgcd in Drosophilanegatively affects their walking activity.

A monitor designed to test walking activity (Trikinetics, Waltham,Mass.) was used to record movement, measured as infrared beam breaks,over a 24 hour period on individual Drosophila. Normally Drosophiladisplay a marked spike in activity at dawn and dusk, irrespective ofgenotype. The data are shown in FIG. 9. To assess basal activity, datawas analyzed from midnight to 8 AM, boxed as region of interest in leftpanel of FIG. 9. Wildtype flies have significantly more activity,measured as infrared beam breaks, than Sgcd⁸⁴⁰ flies which lackγ-sarcoglycan and serve as a model for Limb Girdle Muscular Dystrophytype 2C. This decline in activity mirrors what is seen in musculardystrophy patients who display reduced ambulation due to muscleweakness.

Expression of Mini-Sgcg in Sgcd⁸⁴⁰ Flies Significantly ImprovedNocturnal Activity

FIG. 10 shows that expression of mini-Sgcg in Sgcd⁸⁴⁰ fliessignificantly improved nocturnal activity of mutant Drosophila, measuredas infrared beam breaks (compare Sgcd⁸⁴⁰ vs Scgd⁸⁴⁰, mini-Sgcg in leftpanel). These data indicate that mini-Sgcg can function in the place offull length γ/δ-sarcoglycan which is deleted in Sgcd⁸⁴⁰ flies. The righthand panel shows that nocturnal activity was equally rescued by Sgcg,which is the full length mouse γ-sarcoglycan protein, and mini Sgcg(Sgcg⁸⁴⁰, sgcg compared to Sgcd⁸⁴⁰, mini-Sgcg). These data indicate thatmini-Sgcg is as functional as full length Sgcg for restoring walkingactivity.

Example 5 Mini-Sgcg Protein is Stably Produced in Mammalian SkeletalMuscle and Heart

A transgene using the desmin promoter to drive expression of Mini-Sgcgwas introduced in normal wildtype mice (Tg+). The data are presented inFIG. 11. Skeletal (quadriceps) muscle isolation and immunoblotting wasperformed as described in Hack et al. [J Cell Sci. 113: 2535-44 (2000)].Mini-Sgcg protein is robustly detected in skeletal muscle and heart atthe expected molecular weight of 18 KDa (FIG. 11, arrow). It is notdetected in other cell types such as liver, spleen and kidney. Thisexpression pattern reflects the desmin promoter which drives expressiononly in heart and muscle. Mini-Sgcg is not detected in wildtype,non-transgenic mice (Tg−) (see FIG. 11). The Xpress epitope (Invitrogen)was placed on Mini-Sgcg, and an affinity purified polyclonal rabbitantibody raised to the Xpress epitope was used at a 1:1000 dilution todetect expression from the transgene. These data demonstrate thatmini-Sgcg protein is stable in mammalian muscle and heart, and furtherthat the protein is able to correctly translocate to the musclemembrane.

Characterization of Transgenic Mice

Two different transgenic lines were established expressing mini-Sgcg.The transgenic animals were created using standard protocols. Asdepicted in FIG. 12, line 50 expresses at higher levels than line 84.The upper panel of FIG. 12 shows expression at three differentconcentrations from line 50 or line 84. These data demonstrate thatmini-Sgcg is a stable protein in skeletal muscle and cardiac muscle. Anantibody to endogenous γ-sarcoglycan protein was used to demonstrateexpression of Sgcg protein (full length) in these same samples. Apolyclonal affinity purified rabbit anti-Xpress was generated at PoconoRabbit Farms. It was used at a dilution of 1:1000 for immunoblotting todetect mini-Sgcg (FIG. 12, upper panels). The antibody to γ-sarcoglycanwas previously described [McNally et al., Hum Mol Genet. 5: 1841-7(1996)] and was used to detect endogenous γ-sarcoglycan at 1:1000.

Mini-Sgcg Protein Localizes to the Plasma Membrane of Skeletal Muscle

FIG. 13 shows that mini-Sgcg protein localizes to the plasma membrane ofskeletal muscle when expressed in wildtype normal mice (Tg+). Ananti-Xpress antibody was used to detect expression at the periphery ofeach myofiber consistent with localization at the plasma membrane, orsarcolemma, of skeletal muscle. This intracellular pattern is identicalto normal γ-sarcoglycan (Sgcg) protein, and indicates that mini-Sgcgtranslocates properly. This same signal was not detected in transgenicnegative (Tg−) muscle (FIG. 13, right panel) demonstrating that thissignal derives from the transgene. Immunostaining was performed asdescribed in Hack et al. [J Cell Sci. 113: 2535-44 (2000)]. The affinitypurified polyclonal antibody to the Xpress epitope was used at 1:200.

Expression of Full Length Endogenous γ-Sarcoglycan Protein is Diminishedat the Plasma Membrane when Mini-Sgcg is Present

FIG. 14 shows the results of an experiment designed to test whether themini-Sgcg protein can compete with the endogenous γ-sarcoglycan proteinin vivo. Note that the signal intensity for full length endogenous Sgcgis reduced in the left panel of FIG. 14 compared to the right panel.These data demonstrate that mini-Sgcg competes with endogenous normalSgcg and therefore mini-Sgcg can replace full length Sgcg.Immunostaining was performed as described in Hack et al. [J Cell Sci.113: 2535-44 (2000)]. The polyclonal anti-γ-sarcoglycan antibody wasused at 1:200.

FIG. 15 depicts a model for sarcoglycan assembly. Published literaturedescribes the assembly of the sarcoglycan complex [Chan et al., J CellBiol. 143: 2033-44 (1998); Chen et al., Exp Cell Res. 312: 1610-25(2006); Hack et al., J Cell Sci. 113: 2535-44 (2000)]. In mammalianmuscle, where there are four sarcoglycan subunits, α-, β-, γ-, andδ-sarcoglycan, the β-sarcoglycan and δ-sarcoglycan subunits assemblefirst as a unit in the endoplasmic reticulum (ER)/Golgi apparatus. Thisstep is followed by the addition of α-sarcoglycan and γ-sarcoglycan. Theassembly of the sarcoglycan complex is necessary but not sufficient fortranslocation to the plasma membrane [Chen et al., Exp Cell Res. 312:1610-25 (2006)]. Mutations in sarcoglycan subunits disrupt normaltranslation of the sarcoglycan complex from the ER/Golgi to the plasmamembrane. Translocation to the plasma membrane requires an interactionwith dystrophin and is associated with stabilization of the plasmamembrane. Sarcoglycan complexes containing γ-sarcoglycan (γ) orMini-Sgcg (mγ) successfully translocate to the plasma membrane. InDrosophila muscle, where there is only a single γ/δ-sarcoglycan subunit,mammalian mini-Sgcg can rescue the loss of the single γ/δ-sarcoglycanmoiety and rescue defective heart and muscle function (as shown herein).

Mini-Sgcg Enriches in the Heavy Microsomal Fraction of Muscle

Muscle can be fractionated into a cytoplasmic (C) and a membranefraction. The membrane fraction can be further subdivided into light (L)and (H) microsomes. The sarcoglycan complex is normally found in theheavy microsomal complex containing plasma membrane. Muscle from miceexpressing mini-Sgcg (T+) was fractionated to separate the cytoplasmfrom the light and heavy microsomal fractions. The heavy microsomalfraction contains the ER, Golgi and plasma membrane fractions. In thetwo different transgenic lines (84 and 50), mini-Sgcg enriches greatlyin the heavy microsomal fraction (FIG. 16). This demonstrates thatmini-Sgcg is found in the proper intracellular fraction. The method forisolating microsomes was as previously described [Ohlendieck et al., JCell Biol. 112: 135-48 (1991)] with modification described in [Duclos etal., J Cell Biol. 142: 1461-71 (1998); Hack et al., J Cell Sci. 113:2535-44 (2000)]. The antibody used in the upper panels of FIG. 16 is theaffinity purified anti-Xpress epitope. The antibody used in the lowerpanel of FIG. 16 was a rabbit polyclonal anti-γ-sarcoglycan antibody[McNally et al., Hum Mol Genet. 5: 1841-7 (1996)]. Antibodies were usedat 1:500. All secondary antibodies were from Jackson Immunochemicals(goat-anti-rabbit-HRP) used at 1:1000.

Establishment of Human Cell Lines with SGCG Mutations

Two human cell lines with SGCG mutations have been established for thepurposes of testing exon skipping for the production of mini-Sgcg. Theselines are derived from dermal fibroblasts isolated from human patientswith primary SGCG gene mutations. The top row of FIG. 17 shows cellsfrom an LGMD 2C patient whose disease arises from a mutation deletingexon 7 in SGCG. The bottom row of FIG. 17 shows cells from an LGMD 2Cpatient who is deleted for exon 6 of SGCG. These cell lines wereinfected with retroviruses expressing telomerase and MyoD. The infectionwith the telomerase virus provides an immortalized cell line, and theinfection with the MyoD virus provides a regulated means of inducingmuscle differentiation since the nuclear position of MyoD is under thecontrol of tamoxifen [Kimura et al., Hum Mol Genet. 17: 2507-17 (2008);Kendall et al., Sci Transl Med. 4: 164ra160 (2012)]. This combinationcreates cell lines that are immortalized with telomerase, which providesa ready supply of cells. The regulatable control of MyoD nuclearexpression provides a mechanism by which muscle conversion can beinduced at will. These cell lines serve as cellular models of LGMD 2C,and provide the format in which induced expression of mini-Sgcg can betested in a human cell context. With the addition of tamoxifen, the MyoDlocalizes in the nucleus and the cells undergo differentiation intoelongated myotubes (FIG. 17, middle and right panel).

LGMD 2C Cell Lines Differentiated into the Muscle Lineage

FIG. 18 shows that LGMD 2C cell lines differentiated into the musclelineage following 6 days of differentiation. With the addition oftamoxifen, the LGMD 2C cell line lines undergo morphological changesbecoming elongated and expressing myogenic markers and demonstratingdifferentiation into the muscle lineage. MyoD, a muscle marker, isexpressed from the retrovirus (FIG. 18, middle panel) and desmin, amuscle marker, is induced from MyoD (FIG. 18, right panel) indicatingthat these cells are viable models for muscle disease. The left handpanel of FIG. 18 (Hoechst) shows nuclei. Methods for cell culture aredescribed in Kendall et al. [Sci Transl Med. 4: 164ra160 (2012)]. Theanti-desmin and anti-MyoD antibodies were from ThermoFisher (PA5-17182and MA1-41017, respectively). Secondary antibodies were from MolecularProbes/Invitrogen (goat-anti mouse Cy3, goat anti mouse Alexa488,respectively).

What is claimed is:
 1. An isolated antisense polynucleotide wherein thepolynucleotide specifically hybridizes to an exon target region of agamma sarcoglycan RNA, wherein the exon is selected from the groupconsisting of exon 4 (SEQ ID NO: 1), exon 5 (SEQ ID NO: 2), exon 6 (SEQID NO: 3), exon 7 (SEQ ID NO: 4) and a combination thereof.
 2. Theantisense polynucleotides of claim 1, wherein the polynucleotide cannotform an RNase H substrate.
 3. The antisense polynucleotide of claim 1 orclaim 2, comprising a modified polynucleotide backbone.
 4. The antisensepolynucleotide of any one of claims 1-3, wherein the modifiedpolynucleotide backbone comprises a modified moiety substituted for thesugar of at least one of the polynucleotides.
 5. The antisensepolynucleotide of claim 5, wherein the modified moiety is a Morpholino.6. The antisense polynucleotide of any one of claims 3-5, wherein themodified polynucleotide backbone of at least one of the polynucleotidescomprises at least one modified internucleotide linkage.
 7. Theantisense polynucleotide of claim 6, wherein the modifiedinternucleotide linkage comprises a modified phosphate.
 8. The antisensepolynucleotide of claim 7, wherein the modified phosphate is selectedfrom the group consisting of a methyl phosphonate, a methylphosphorothioate, a phosphoromorpholidate, a phosphoropiperazidate and aphosphoroamidate.
 9. The antisense polynucleotide of any one of claims3-8, wherein the polynucleotide is a 2′-O-methyl-oligoribonucleotide.10. The antisense polynucleotide of any one of claims 1-9, wherein thepolynucleotide is chemically linked to one or more conjugates thatenhance the activity, cellular distribution, or cellular uptake of theantisense polynucleotide.
 11. A pharmaceutical composition, comprisingthe antisense polynucleotide of any one of claims 1-10 and aphysiologically compatible phosphate buffer.
 12. A kit comprising theantisense polynucleotide of any one of claims 1-10, optionally in acontainer, and a package insert, package label, instructions or otherlabeling.